aqueous solutions of the sample (up to ~100°C). It is likely that removal of hydration water destabilizes the N-ethylpyridinium moieties toward thermal decomposition. Thus, MnTE-2-PyPCl5 should not be taken to absolute dryness. Heating of the solid to temperatures above 44°C at 1 atm may change its composition (by loss of water molecules), but with no effect to the intrinsic catalytic activity (per Mn). Heating of MnTE-2-PyPCl5 above 150 °C may be accompanied by a decrease (or complete loss) of catalytic activity as the removal of ethyl groups is followed by a decrease of both Mn(III)/Mn(II) reduction potential and electrostatic facilitation for O2

•– dismutation/ONOO–

decomposition. These results are consistent with our earlier assumption (Rebouças et al. J. Pharm. Biomed. Chem. 2008, 48, 1046) that the origin of the mixture of partially alkylated compounds in the commercial MnTE-2-PyPCl5 samples and their methyl analogues may likely relate to inappropriate conditions of drying and/or handling of the samples, prior to arrival at the researchers hands. CAPES, CNPq, UFPB, NIH U19AI067798

613 Kinetic Analysis of Endothelial Nitric Oxide Synthase­Catalyzed Decomposition of Peroxynitrite Natalia Romero1, Natalia Subelzú1, and Rafael Radi1 1CFRBM, Fac. de Medicina, Universidad de la República, Uruguay Nitric oxide (•NO) is synthesized in endothelial cells by the endothelial isoform of nitric oxide synthase (eNOS), an homodimeric-enzyme with a 4Cys-Zn cluster that utilizes L-arginine, O2 and NADPH as substrates and heme, FAD, FMN, Ca2+/calmodulin and tetrahydrobiopterin (BH4) as cofactors. Several physiopathological conditions such as diabetes or hypertension are associated with increased vascular superoxide radical (O2

•–) production that reacts fast with •NO decreasing its bioavailability, a phenomenon called endothelial dysfunction. In addition, the product of the reaction yields the strong oxidant peroxynitrite, which reacts fast with cellular targets provoking the loss- or gain- of-function. Previous studies suggested that peroxynitrite oxidizes eNOS and, as a consequence, the enzyme becomes “uncoupled”, i.e., produces O2

•– instead of •NO as the reaction product. Herein, we have investigated the mechanism of reaction of peroxynitrite with the recombinant oxygenase domain of eNOS (oxy-eNOS). Oxy-eNOS was obtained in E.Coli BL21 co-transformed with calmodulin and the purified enzyme was mainly present in its dimeric form as it was confirmed by gel filtration and low temperature SDS-PAGE. Fast UV-Vis spectra of the enzyme did not show any change after peroxynitrite addition. However, fast-kinetic studies following peroxynitrite decomposition at 302 nm demonstrated that the enzyme catalyzes peroxynitrite decomposition with a second order rate constant of ~ 2.5 x 106 M-

1s-1 at 37°C and pH 7.4. The reaction is inhibited (~ 10 times) in the presence of the substrate L-arginine but it does not depend on the presence of BH4, that only reacts with peroxynitrite derived-radicals as was also observed by fast-kinetic analysis. Measurement of reaction products indicates that NO3

– is formed as the final product of peroxynitrite decomposition in the presence of the enzyme. Peroxynitrite-dependent nitration of the tyrosine analogue p-hydroxy-phenyl acetic acid was not promoted in the presence of subestoicheometric amounts of the enzyme respect to peroxynitrite suggesting isomerization of peroxynitrite as the reaction mechanism. The results obtained herein demonstrate that eNOS can represent a biologically-relevant target of intracellularly-generated peroxynitrite and will contribute in the design of new scavengers that catalytically decompose peroxynitrite.

614 Mitochondrial Oxygen Affinity Predicts Basal Metabolic Rate in Humans Tomas A Schiffer1, Filip Jon Larsen1,2, Björn Ekblom1,2, Eddie Weitzberg1, and Jon O Lundberg1 1Karolinska Institute, 2Swedish School of Sports and Exercise Science Large individual variations in basal metabolic rate (BMR) exist, differences that remain even when correcting for fat free body mass. The cellular mechanism behind this is a fundamental problem in biology. The hypothesis that “thrifty genes” exist has been proposed, genes that make certain individuals energetically efficient with low BMR, increased fat deposition and increased susceptibility for obesity and diabetes. However, mechanistic insights are still lacking and no convincing functional, cellular or mitochondrial characteristics have been found that can explain the large differences in BMR. In this study we assessed mitochondrial efficiency and mitochondrial oxygen affinity (p50mito) by high resolution respirometry in muscle biopsies from 13 healthy young males with a large inter-individual variation in BMR (range 2.4-4.2 ml O2 kg-1 min-1). This was combined with measurements of metabolic efficiency and aerobic power on a cycle ergometer. p50mito was negatively associated (R2=0.52, p=0.007) with mass-specific BMR, oxygen cost during exercise at 50% of VO2 peak (R2=0.57, p=0.005) and maximal aerobic power (R2=0.34, p=0.03). There were no correlations between any measure of metabolic efficiency and the mitochondrial efficiency assessed as the mechanistic P/O ratio. To our knowledge this is the first time an intrinsic mitochondrial characteristic has been associated with whole body metabolic efficiency in vivo. These results also imply that maximum aerobic efficiency and power are mutually exclusive. Efficient metabolism and high p50mito means low mitochondrial affinity for oxygen which comes with the tradeoff of increasing oxygen limitation at low oxygen tensions such as those during strenuous exercise.

615 New Mechanism for the Oxidation of Hydroxycinnamic Acid Derivatives: Regeneration of the Molecules in Oxygenated Condition Soumyakanti Adhikari1, Shashi P Shukla1, and Tulsi Mukherjee1 1Bhabha Atomic Research Centre Free radical scavenging is one of the accepted mechanisms for the antioxidant action of hydroxycinnamic acids (HCAs). In last few decades copious experimental data have been reported in the literature on the effectiveness of these chemical systems as radical scavengers. Many theoretical studies have corroborated experimental data for better understanding of the antioxidant pathway. Phenols are known to act as free radical scavengers either by direct hydrogen transfer (HAT) or by sequential proton loss electron transfer (SPLET) from the phenol moiety. The overall change in the molecule is the same in both mechanisms as far as the end-products is concerned. In most of the studies reported so far, the role of the unsaturation in the side chain of the phenolic acids have been accounted for the enhanced stability of the phenoxyl radical via conjugation. However, in the present study we have observed that an electron transfer from the double bond can take place at the initial step in the free radical scavenging process under certain condition. This reaction pathway had not been reported or discussed in earlier studies. In the reaction of 1-hydroxyethyl radical (HER) with HCAs at natural pH, a radical is formed, which is neither an adduct nor the phenoxyl radical. The rate constants for this reaction range from 1x109 to 5x109 dm3 mol-1 s-1 for cinnamic acid, caffeic acid, ferrulic acid and coumaric acids. A similar result has also been

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